Project Summary The clinical use of immune checkpoint inhibitors (ICIs) that target the programmed cell death protein (PD-1) and its ligand PD-L1 have significantly improved outcomes in patients with numerous cancer types. However, a large proportion of patients eventually relapse on therapy. Currently, preclinical studies of acquired resistance to ICIs have been limited because most tumor models do not faithfully mimic treatment failure as it occurs in patients. This gap has limited the understanding of resistance mechanisms that are urgently needed to identify optimal second-line treatments. In several cancer types, receptor tyrosine kinase inhibitors (RTKIs) that target metastasis and angiogenesis pathways are being tested for efficacy after anti-PD-L1 (?PD-L1) treatment failure. Currently, there is no method to identify who will respond. Here, we propose that intracellular functions of PD-L1 may be the key to determining RTKI responses after ICI failure. PD-L1 has cytoplasmic domains that can control various signaling pathways but these functions have not been tested in models of resistance. We have preliminary data that suggest ?PD-L1 treatment failure can induce secretory changes controlled by IFN? which, in turn are regulated by intracellular PD-L1 signaling. Notably, we also found that PD-L1 treatment can confer a cross-resistance to RTKIs. The overall hypothesis of this research is that resistance to PD-L1 inhibition can enhance IFN?-mediated secretory profiles through intracellular PD-L1 signaling which, in turn, can reduce the efficacy of RTKIs. To test this, we first propose to investigate the effects of chronic PD-L1 treatment on intracellular functions regulating IFN-? mediated secretory changes (Aim 1). This work will be conducted using novel mouse tumor models of acquired ?PD-L1 treatment resistance developed in our laboratory. Next, we will test whether IFN? signaling inhibition may improve the efficacy of RTKI treatments (including cabozantinib and axitinib) in ?PD-L1 treatment-refractory models (Aim 2). Together, these studies aim to identify a novel role for IFN? signaling in ?PD-L1 treated tumor cells with the goal of improving second- line treatment options in patients.